WO2000029437A1 - VARIANTS HIF-1α DU FACTEUR INDUCTIBLE PAR L'HYPOXIE (HIF), LEURS METHODES D'IDENTIFICATION, ET LEURS MODULATEURS - Google Patents

VARIANTS HIF-1α DU FACTEUR INDUCTIBLE PAR L'HYPOXIE (HIF), LEURS METHODES D'IDENTIFICATION, ET LEURS MODULATEURS Download PDF

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WO2000029437A1
WO2000029437A1 PCT/SE1999/002053 SE9902053W WO0029437A1 WO 2000029437 A1 WO2000029437 A1 WO 2000029437A1 SE 9902053 W SE9902053 W SE 9902053W WO 0029437 A1 WO0029437 A1 WO 0029437A1
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seq
human
domain
variant
cells
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PCT/SE1999/002053
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Anders Berkenstam
Lorenz Poellinger
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Pharmacia Ab
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Priority to JP2000582422A priority Critical patent/JP2002530063A/ja
Priority to EP99958594A priority patent/EP1141005B1/fr
Priority to CA002350830A priority patent/CA2350830A1/fr
Priority to NZ511450A priority patent/NZ511450A/en
Priority to DE69937199T priority patent/DE69937199D1/de
Priority to BR9915263-0A priority patent/BR9915263A/pt
Priority to AU15931/00A priority patent/AU761594B2/en
Publication of WO2000029437A1 publication Critical patent/WO2000029437A1/fr
Priority to HK02105080.8A priority patent/HK1043372B/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel variants of human hypoxia-inducible factor (HLF)- l ⁇ , as well as to the use of the said variants in screening assays for the elucidation of functional motifs in HLF-l ⁇ and for the identification of compounds that modulate the function of HIF-l ⁇ , said compounds being potentially useful in the regulation of target genes normally associated with HIF-l ⁇ such as genes involved in angiogenesis, erythropoiesis, and glycolysis.
  • HIF hypoxia-inducible factor
  • these cells have developed ways to sense alterations in oxygen levels and, during this process, acquired the ability to conditionally modulate the expression of genes involved in adaptive physiological responses to hypoxia including angiogenesis, erythropoiesis, and glycolysis.
  • genes involved in adaptive physiological responses to hypoxia including angiogenesis, erythropoiesis, and glycolysis.
  • These genes include vascular endothelial growth factor, eryhtropoietin, several glycolytic enzymes and inducible nitric oxide synthase, and have all been shown to contain hypoxia responsive elements (HREs) (for reviews, see Guillemin and Krasnow (1997) Cell 89, 9-12; Wenger and Gassmann (1997) Biol. Chem. 378, 609-616) .
  • HREs hypoxia responsive elements
  • hypoxia inducible factor HIF
  • Arnt the hypoxia inducible factor
  • Both these transcription factors belong to the rapidly growing family of basic-helix-loop-helix (bHLH)-PAS (Per, Arnt, Sim) proteins.
  • bHLH/PAS transcription factors play diverse biological roles.
  • HTF-l ⁇ - and Arnt-deficient embryonal stem cells and mice have indicated critical roles of both these factors in cardiovascular development and regulation of HRE-driven target genes (Maltepe et al. (1997) Nature 386, 403-407).
  • the mechanism of hypoxia-dependent formation and activation of the HLF-l ⁇ -Arnt complex is presently poorly understood.
  • the present inventors and others have recently demonstrated that HEF-lc protein levels are specifically and massively upregulated under hypoxic conditions in most if not in all cells. Since HIF-l ⁇ mRNA levels are unaltered in response to hypoxia, this mode of regulation appears to occur via a post-transcriptional step involving stabilization of HUF-l ⁇ protein levels (Huang et al.
  • Import of transcription factors into the nucleus is frequently a conditionally regulated process that occurs in response to various external and internal stimuli as well as to a developmental cues (Vandromme et al. (1996) Trends Biochem. Sci. 21, 59-64). Thus, this process can constitute a critical mechanism of regulation of transcription factor activity. Active, energy-dependent transport of proteins to the nucleus requires the presence of one or several nuclear localization signals (NLSs) within the transported protein (or its interaction partner).
  • NLSs nuclear localization signals
  • NLS motifs are short amino acid moieties that can be divided into two main groups: (i) the SV40 large T antigen type of NLS, characterized by a single cluster of four or more consecutive basic residues; and (ii) a bipartite NLS, consisting of two basic residues, a spacer of any ten amino acids, and a basic cluster where three of the next five residues are basic (Vandromme et al., supra, and references therein).
  • HIF-l ⁇ its amino acid sequence and polynucleotide sequence are disclosed by Wang et al. (1995) Proc. Natl. Acad. Sci. USA 92, 5510-5514 and in WO 96/39426. It has been suggested (Jiang et al. (1997) J. Biol. Chem. 272, 19253-19260) that amino acids 531-826 of HLF-l ⁇ contain two transactivation domains, TAD-N (amino acids 531-575) and TAD-C (amino acids 786-826). Further, Pugh et al. ((1997) J. Biol. Chem.
  • HLF-la amino acids 549-582 and 775-826
  • HLF-l ⁇ shows hypoxia- inducible nuclear translocation.
  • COS7 cells were transiently transfected with pCMN-HIF-l ⁇ and after 24 h expression incubated for 6 additional h under either normoxic (21% O2) or hypoxic (1% O2) conditions before fixation for immunocytochemistry. Localization of expressed HIF-l ⁇ was determined by indirect immunofluorescence using anti-HIF-l ⁇ antiserum as described in Materials and Methods.
  • GFP-HLF-l ⁇ Hypoxic regulation of nuclear translocation by GFP-HLF-l ⁇ .
  • A Schematic representation showing the GFP and GFP-HLF-l ⁇ protein constructs. For HLF-l ⁇ , location of the basic helix-loop-helix and PAS (Per/Arnt/Sim) domains is also indicated.
  • B Hypoxia and hypoxia-mimicking chemicals induce rapid nuclear translocation of GFP-HEF-l ⁇ fusion proteins.
  • COS7 cells were transiently transfected with either GFP or of GFP-HUF-l ⁇ expression vectors and after 24 h expression induced for 6 h either with 1 % O2, 100 ⁇ M C0CI2 or with 100 ⁇ M 2,2'-dipyridyl (DP) before microscopy.
  • DP 2,2'-dipyridyl
  • COS7 cells were transfected with nonfusion GFP expression vector (lanes 1 and 2) or with GFP-HLF-l (lanes 3 and 4). Following transfection cells were exposed to 21% or 1% (hypoxia) oxygen for 30 h as indicated by - and + signs.
  • Whole cell extracts were prepared as described in Materials and Methods, and 50 ⁇ g aliquots were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting using anti-HEF-l ⁇ antiserum.
  • GFP-HLF-l ⁇ protein is indicated by an arrowhead, whereas the asterisk denotes endogenous HLF-l ⁇ immunoreactivity.
  • the positions of the molecular weight markers (in kDa) are indicated on the left.
  • D Time- dependent nuclear translocation of GFP-HIF- l ⁇ . COS7 cells were transfected as in panel A with GFP-HIF- l ⁇ expression vectors and induced with 100 ⁇ M 2,2'-dipyridyl. Photographs were taken at the indicated timepoints after induction.
  • E Graph presentation of the nuclear entry of GFP-fflF-l ⁇ .
  • N is the percentage of cells showing exclusively nuclear fluorescence.
  • FIG. 3 Nuclear export of HIF-l ⁇ upon withdrawal of hypoxic signal.
  • COS7 cells were transfected with the GFP-HLF-l ⁇ expression vector (6 ⁇ g/60-mm dish) for 6 h and grown under atmospheric oxygen for 12, whereafter they were either induced with 100 ⁇ M 2,2'-dipyridyl or left uninduced for 1 h. Subsequently 2,2'- dipyridyl was withdrawn by change of medium and washing of the cells, and the cells were therefter incubated in the absence (-) or presence (+) of 25 ⁇ M cycloheximide for 24 h. During this time period cells were observed microscopically at different timepoints as indicated by arrows.
  • HIF-l ⁇ is redistributed from the nucleus upon withdrawal of 2,2'-dipyridyl.
  • the subcellular distribution of GFP-HIF- l ⁇ is shown at different timepoints following withdrawal. The percentage of cells belonging to each category in indicated (for classification, see Table I).
  • Table I For reference the intracellular distribution of GFP-HIF- l ⁇ prior to induction with 2,2'- dipyridyl is also shown.
  • FIG. 4 Subcellular distribution of GFP-HIF- 1 ⁇ fusion proteins.
  • A Schematic presentation of fusion proteins. All expression constructs were assembled into pCMX-SAH/Y145F vector expressing a humanized GFP under the control of the CMV promoter.
  • B Subcellular distribution of GFP-HIF- l ⁇ chimeric proteins. Fusion constructs corresponding to those presented panel A were transfected into COS7 cells and after 24 h expression either 100 ⁇ M 2,2'-dipyridyl (DP) or vehicle (H2O) were added to the culture medium and incubated for 6 h before observation. Photographs were taken using a Zeiss fluorescent microscope.
  • DP 2,2'-dipyridyl
  • H2O vehicle
  • COS7 cells were transfected with various GFP- HIF-l ⁇ fusion protein expression plasmids carrying either wild-type or mutated HLF-l ⁇ NLS sequences.
  • the mutations were either single (Arg to Ala) or double (Arg to Gly) amino acid exchanges at residues 17 and 18, or a single amino acid exchange (Lys to Thr) at codon 719, respectively.
  • cells were treated with 100 ⁇ M 2,2'-dipyridyl or with vehicle only for 6 h before fluorescence microscopy.
  • COS7 cells were cotransfected either with GFP or GFP-HIF- l ⁇ expression vectors and a hypoxia responsive reporter gene (HRE-luc in the presence of either wild-type Arnt or a dominant negative Arnt mutant, Arnt ⁇ b, lacking the DNA binding b domain.
  • HRE-luc hypoxia responsive reporter gene
  • Cells were exposed for 21% or 1% O 2 for 30 h before harvest and reporter gene assays. Normalized reporter gene activities are expressed relative to that of nonfusion GFP in normoxia. The results of two independent experiments performed in triplicates +/- SEM are shown.
  • C Functional analysis of GAL4-HIF-l ⁇ fusion proteins.
  • COS7 cells were transiently transfected with pCMX- GAL4-HLF-la and after 24 h expression exposed to normoxic (21% O 2 ) or hypoxic (1% O 2 ) conditions for 6h before immunocytochemistry.
  • Indirect immunofluorescence of expressed GAL4-HLF-l ⁇ fusion protein was conducted with anti-GAL4-DBD antiserum, biotinylated anti-rabbit Ig antibody, and Texas Red conjugated with streptoavidin.
  • NLS mutation eliminating inducible nuclear import does not affect hypoxia dependent recruitment of CBP.
  • GAL4-HIF-l ⁇ or GAL4-HEF-l ⁇ transactivation domain fusion proteins were transfected into COS7 cells together with a GAL4 responsive reporter plasmid with or without CBP expression plasmid. Cells were exposed for 21% or 1% O2 for 30 h before harvest and reporter gene assays.
  • C The C-terminal transactivation domain of HIF-l ⁇ is targeted by CBP.
  • GAL4 fusion protein containing the minimal C-terminal transactivation domain (amino acids 776-826) or a deletion mutant thereof were transfected into COS7 cells together with a GAL4 responsive reporter plasmid in the absence or presence of the CBP expression vector. Cells were exposed for 21% or 1% O 2 for 30 h before harvest and reporter gene assays. After normalization for transfection efficiency using alkaline phosphatase activity, reporter gene activities are expressed relative to that of GAL4 in normoxia.
  • Fig. 8 The transcriptional coactivator TLF2 enhances the HIF-l ⁇ -mediated transactivation function and interacts with HIF-l ⁇ in vivo.
  • COS7 cells were transiently cotransfected as described in Materials and Methods with 0.5 ⁇ g pT81/ ⁇ RE-Luc reporter construct, hHLFl- ⁇ expression vector (pCMV4/HIF-l ⁇ , 0.2 ⁇ g), Arnt expression vector (pCMV4/Arnt, 0.2 ⁇ g) and 0.75-1.5 ⁇ g of either ⁇ F2 (pSG5/TIF2) or CBP (Rc/RSV-CBP-HA), as indicated.
  • the total amount of DNA was kept constant by the addition of parental pCMV4 when appropriate.
  • Luciferase activities were normalized for transfection efficiency by cotransfection of alkaline phosphatase expressing pRSV-AF. Data are presented as luciferase activity relative to cells transfected with pCMN4 and reporter gene only and incubated at normoxia. Values represent the mean ⁇ S.E. of two independent experiments.
  • the transcriptional coactivator SRC-1 stimulates HIF-l ⁇ activity in a hypoxia-dependent manner and interacts in vitro with HIF-l ⁇ .
  • COS7 and B human embryonic kidney 293 cells were cotransfected with pT81/HRE-Luc (0.5 ⁇ g), 0.2 ⁇ g of pCMV4 HIF-lcc (hHFl- ⁇ ), 0.2 ⁇ g of pCMV4/Arnt (Arnt), and 0.75-1.5 ⁇ g of SRC-1 (pSG5/SRC-l), as indicated.
  • Six h after transfection cells were exposed to either 21 or 1%> O 2 for 36 h before harvest.
  • pGAL4/fflF 71-826 was cotransfected into COS7 cells together with a reporter plasmid expressing the luciferase gene driven by the thymidine kinase minimal promoter under the control of five copies of GAL4- binding sites, and 1.5 ⁇ g of either full-length SRC-1 or a deletion mutant of SRC-1, SRC- 1 ⁇ PAS, lacking the PAS domain.
  • Cells were exposed to 21 or 1% O 2 for 36 h before harvest and reporter gene assays. Luciferase values are presented as relative luciferase activity as described in the legend to Fig. 1. The results of two independent experiments performed in duplicate ⁇ S.E. are shown.
  • LXXLL motifs of SRC-1 are not required for HF-l ⁇ -mediated transactivation function and interaction with HF-l ⁇ in vivo.
  • pGAL4/HF 71-826 was cotransfected into COS7 cells together with a GAL4-responsive reporter plasmid, and 1.5 ⁇ g of either full-length SRC-1 or mutated SRC-1, SRC-1 M1234.
  • Luciferase values are presented as relative luciferase activity as described in the legend to Fig. 1. The results of two independent experiments performed in duplicate ⁇ S.E. are shown.
  • Fig. 11 Definition of HF-l ⁇ structures which are regulated by the CBP and SRC-1 coactivators.
  • A Different subregions of HF-l ⁇ were fused to the GAL4 DBD and transfected into COS7 cells together with a GAL4-responsive reporter plasmid in the absence or presence of 1.5 ⁇ g of either CBP or SRC-1 expression plasmids. Cells were exposed to 21 or 1% O 2 for 36 h before harvest.
  • B The C-terminal transactivation domain of Arnt mediates transcriptional activation by CBP and SRC-1.
  • COS 7 cells were cotransfected with the indicated GAL4/Arnt fusion proteins and 1.5 ⁇ g of CBP or SRC-1 together with a GAL4-responsive reporter plasmid. Six h after transfection cells were exposed to either 21 or 1% O for 36 h before harvest and reporter gene assays.
  • C HF-l ⁇ prevents Arnt from functionally interacting with CBP and SRC-1.
  • GAL4/Arnt 128-774 and a deletion mutant (GAL4/Arnt 128-603) were transfected into COS7 cells together with 0.2 ⁇ g hHF-l ⁇ expression vector and 1.5 ⁇ g of CBP and SRC-1, as indicated.
  • Luciferase activity was measured following 36 h of exposure to either 21 or 1% O 2 . Normalized reporter gene activities are expressed relative to that of non- fusion GAL4 in normoxia. The results of three independent experiments performed in duplicate ⁇ S.E. are shown.
  • CBP and SRC-1 cooperatively enhance HF-l ⁇ -mediated transcriptional activation.
  • A COS7 cells were transiently cotransfected with pCMV4/HF-l ⁇ and 0.75-1.5 ⁇ g of SRC-1 and/or 0.75-1.5 ⁇ g of CBP expression plasmids together with a hypoxia-responsive reporter gene (pT81/HRE-Luc) and subsequently exposed to 21 or 1% O 2 . Luciferase values are presented as relative luciferase activity as described in the legend to Fig. 1. The results of three independent experiments performed in duplicate ⁇ S.E. are shown.
  • B Cooperative activation by CBP and SRC-1 of GAL4/HF-l ⁇ fusion proteins.
  • COS7 cells were cotransfected with GAL4/HF-l ⁇ fusion constructs together with a GAL4-responsive reporter plasmid in the absence or presence of CBP and/or SRC-1, as indicated (in ⁇ g). Cells were exposed to 21 or 1% O 2 before harvest. After normalization for transfection efficiency using alkaline phosphatase activity, reporter gene activities are expressed as relative to that of GAL4 in normoxia. The results of two independent experiments performed in duplicate ⁇ S.E. are shown. (C) p300 protein stimulates the activity of the two minimal transactivation domains of HF-l ⁇ in a hypoxia-dependent manner.
  • GAL4/HF-l ⁇ fusion constructs together with a GAL4-responsive reporter plasmid in the absence or presence of 1.5 ⁇ g of Ref-1 (pCMV5/Ref-l), as indicated.
  • Cells were exposed to 21 or 1% O 2 before harvest. After normalization for transfection efficiency using alkaline phosphatase activity, reporter gene activities are expressed as relative to that of GAL4 in normoxia. The results of two independent experiments performed in duplicate ⁇ S.E. are shown.
  • (B) Ref-1 potentiates CBP and SRC-1 activation of HF-l ⁇ .
  • the same GAL4/HF- l ⁇ fusion proteins as shown in (A) were cotransfected into COS7 cells together with a
  • GAL4-responsive reporter plasmid in the absence or presence of different combinations of Ref-1 (0.75 ⁇ g), CBP (0.75 ⁇ g), and/or SRC-1 (0.75 ⁇ g), as indicated.
  • the bottom panel shows an enlargement of the area marked with dots.
  • HF-l ⁇ is a multi-step process, which includes hypoxia-dependent nuclear import and activation (derepression) of the transactivation domain, resulting in recruitment of the CBP/p300 coactivator.
  • Inducible nuclear accumulation has been shown to be dependent on a nuclear localization signal (NLS) within the C-terminal end of HF-l ⁇ that also harbors the hypoxia- inducible transactivation domain.
  • NLS nuclear localization signal
  • Nuclear import of HF-l ⁇ was inhibited by either deletion or a single amino acid substitution within the NLS sequence motif and, within the context of the full-length protein, these mutations also resulted in inhibition of the transactivation activity of HF-l ⁇ and recruitment of CBP.
  • nuclear localization er se is not sufficient for transcriptional activation, since fusion of HF-l to the heterologous GAL4 DNA binding domain generated a protein which showed constitutive nuclear localization but required hypoxic stimuli for function as a CBP-dependent transcription factor.
  • the transcriptional coactivators SRC-1 and TF2 also support HF-l ⁇ -dependent transcriptional activation.
  • CBP and the SRC-1/TF2 class of coactivators target both N- and C-terminal transactivation domains of HF-l ⁇ , and are potentiated in their function by the redox regulatory enzyme Ref-1.
  • Ref-1 physically interacts with HF-l ⁇ under hypoxic conditions.
  • hypoxia-inducible nuclear import and transactivation by Ref-1 -dependent recruitment of the transcriptional coactivators CBP, SRC-1/TF2 can be functionally separated from one another and play critical roles in signal transduction by HF-l ⁇ .
  • this invention relates to a method for identifying compounds capable of modulating the function of a functional domain of human HF-l ⁇ , said method comprising
  • transactivator domain located in human HF-l ⁇ essentially between amino acids 531 to 584
  • transactivator domain located in human HF-l ⁇ essentially between amino acids 813 and 826
  • the said method comprises (i) contacting a candidate compound with a cell expressing a said variant of human HF-l ⁇ , said variant being conjugated to a molecular probe, and (ii) detecting the localization of the said molecular probe within the cell.
  • the said molecular probe is preferably a fluorescent probe, such as the Aequeora victoria Green Fluorescent Protein, or a variant thereof. A change in the relative fluorescence of the nucleus to the cytoplasm is then indicative for a compound capable of modulating the function of the said functional domain.
  • the said variant of human HF-l ⁇ is regulating the activity of a reporter gene; a change in the activity of the reporter gene being indicative of a compound capable of modulating the function of the said functional domain.
  • the HF- l ⁇ variant can e.g. be fused to the DNA binding domain of GAL4, and the cells expressing the HF-l ⁇ variant will contain a GAL4 response element operatively linked to a reporter gene.
  • the variant of human HF-l ⁇ can be fused to the Aequeora victoria Green Fluorescent Protein, and the cells expressing the HF-l ⁇ variant could suitably contain an erythropoietin hypoxia response element operatively linked to a reporter gene, e.g. a firefly luciferase gene.
  • the method according to the invention can suitably be carried out in the presence of a transcriptional coactivator, in particular one or more of the transcriptional coactivators designated SRC-1, TF2 or CBP.
  • a transcriptional coactivator in particular one or more of the transcriptional coactivators designated SRC-1, TF2 or CBP.
  • the method according to the invention can suitably be carried out in the further presence of the redox regulatory protein designated Ref- 1.
  • the said variant of human HF-l ⁇ can e.g. be HF-l ⁇ /1-74 (SEQ ID NO: 2); HF-l ⁇ /1-245 (SEQ ID NO: 3); HF-l ⁇ / ⁇ 178-390 (SEQ LD NO: 6); HF-l ⁇ /331-641 (SEQ ID NO: 7); HF-l ⁇ /526- 641 (SEQ ID NO: 8); HF-l ⁇ /526-813 (SEQ ID NO: 9); or HF-l ⁇ /526-826 (SEQ ID NO: 10).
  • the said variant of human HF-l ⁇ can e.g. be HF-l ⁇ 1-74 (SEQ ID NO: 2); HF-l ⁇ /1-245 (SEQ ID NO: 3); HF-l ⁇ /1-330 (SEQ ID NO: 4); HF-l ⁇ /1- 652 (SEQ LD NO: 5); HF-l ⁇ /331-641 (SEQ LD NO: 7); or HF-l ⁇ /526- 41 (SEQ ID NO: 8).
  • the said functional domain of human HF-l ⁇ is the transactivator domain (N-TAD), located in human HF-l ⁇ essentially between amino acids 531 to 584
  • the said variant of human HF-l ⁇ can e.g. be HF-l ⁇ /1-74 (SEQ LD NO: 2); HF-l ⁇ /1-245 (SEQ ID NO: 3); HF-l ⁇ /1-330 (SEQ ID NO: 4); HF-l ⁇ /776-826 (SEQ ID NO: 14); or HF-l ⁇ /776-813 (SEQ LD NO: 15).
  • the said variant of human HF-l ⁇ can e.g. be HF-l ⁇ /1-74 (SEQ ID NO: 2); HF-l ⁇ /1-245 (SEQ ID NO: 3); HF-l ⁇ /1-330 (SEQ ID NO: 4); HF-l ⁇ /1-652 (SEQ ID NO: 5); HF-l ⁇ /331-641 (SEQ ID NO: 7); HF-l ⁇ /526-641 (SEQ LD NO: 8); HF-l ⁇ /526-813 (SEQ TD NO: 9); HF- l ⁇ /1-813 (SEQ TD NO: 12); HF-l ⁇ /531-584 (SEQ D NO: 13); HF-l ⁇ /776-813 (SEQ ID NO: 15).
  • the invention provides an isolated variant of human HF-l ⁇ , said variant being selected from the group consisting of HF-l ⁇ /1-74 (SEQ ID NO: 2); HF- l ⁇ /1-245 (SEQ ID NO: 3); HF-l ⁇ /1-330 (SEQ ID NO: 4); HF-l ⁇ /1-652 (SEQ ID NO: 5); HF-l ⁇ / ⁇ 178-390 (SEQ ID NO: 6); HF-l ⁇ /331-641 (SEQ ID NO: 7); HF-l ⁇ /526- 641 (SEQ ID NO: 8); HF-l ⁇ /526-813 (SEQ ID NO: 9); HF-l ⁇ /526-826 (SEQ ID NO: 10); HF-l ⁇ /71-826 (SEQ ID NO: 11); HF-l ⁇ /1-813 (SEQ ID NO: 12), HF-l ⁇ /531-584 (SEQ ID NO: 13); HF-l ⁇ /776-826 (SEQ ID NO: 14); and HF-l ⁇
  • HIF-l ⁇ shows hypoxia-inducible nuclear import
  • N cells containing exclusively nuclear fluorescence
  • N > C cells in which the nuclear fluorescence dominates over cytoplasmic fluorescence
  • N ⁇ C cells in which the intensity of fluorescence in the cytoplasm exceeds that in the nucleus.
  • HF-l ⁇ contains between aa 17-33 a bipartite NLS motif similar to that of Xenopus laevis nucleoplasmin (Dingwall et al. (1987) EMBO J. 6, 69-74) ( Figure 5 A).
  • Reporter gene activity was not significantly altered by hypoxic treatment (1%> O 2 ; Figure 6A) or incubation with 2,2'-dipyridyl (data not shown) either in the absence of coexpressed proteins or in the presence of transiently expressed parental GFP, most probably due to low levels of endogenous HF-1 activity in COS7 cells.
  • coexpression of GFP-HF-l ⁇ resulted in stimulation (about 2-fold) of reporter gene activity already at normoxia, possibly due to stabilization of HF-l ⁇ levels by the GFP moiety and, as described above, a small but detectable pool of HF-l ⁇ localized in the nucleus under these conditions.
  • This reporter gene activity was further stimulated about 4- fold by hypoxia (Figure 6A).
  • transient expression of wild-type HF- l ⁇ also yielded a similar pattern of reporter gene activation, albeit with a slightly lower potency generating about a 2-fold activation response upon exposure to hypoxia.
  • fusion of GFP to the immediate proximity of the N-terminal basic (DNA binding) region did not interfere with transcriptional activation.
  • stabilization of the fusion protein (Figure 2C) by the GFP moiety may rather have enhanced the potency of the activation response produced by HF-l .
  • GFP-HF-l ⁇ fusion proteins lacking the DNA binding bHLH region did not induce any reporter gene activity.
  • HF-l ⁇ To analyze functional activities of HF-l ⁇ independently of the endogenous DNA binding and dimerization domains and the Arnt partner, we next fused HF-l ⁇ to the heterologous DNA binding domain of GAL4. Functional activity of the fusion protein was monitored in COS 7 cells by a cotransfection assay using a reporter gene construct carrying five GAL4 DNA binding elements in front of thymidine kinase promoter and the luciferase gene. Six hours after transfection media was changed, and cells were either left uninduced or exposed to 1%) oxygen for 30 h prior to harvest. In the presence of either empty expression vector or a vector expressing the minimal GAL4 DNA binding domain, reporter gene activity was unaltered by reduced oxygen levels.
  • the CREB binding protein CBP is a transcriptional coactivator protein known to interact with a number of constitutively active or inducible DNA binding transcription factors including, among others, CREB, c-Fos, c-Jun, various members of the steroid receptor s superfamily, and p53 (Shikama et al. (1997) Trends Cell Biol. 7, 230-236). CBP has also been demonstrated to physically associate with HF-l ⁇ and to play a role in hypoxia- dependent transactivation of erythropoietin promoter in Hep3B cells (Arany et al. (1996) Proc. Natl. Acad. Sci. USA 93, 12969-12973).
  • CBP was also able to dramatically enhance the activity of the GAL4-HF- l ⁇ /526-826 K719T fusion protein carrying a point mutation in the hypoxia-inducible NLS motif of HF-l ⁇ .
  • the effect of CBP to support inducible transcription by either GAL4 HF-l ⁇ /526-826 or GAL4-HF-l ⁇ /526-826 K719T was very similar (Figure 7B), thus demonstrating that inactivation of the hypoxia-inducible NLS motif does not alter the ability of this domain to respond to hypoxia by coactivator recruitment and subsequent transactivation.
  • TF2 belongs to the pi 60 family of coactivators which have been shown to be required for nuclear receptor-mediated transcriptional activation (Takeshita et al. (1997) J. Biol. Chem. 272, 27629-27634). Transient transfection experiments were initially performed using COS7 cells. As shown in Fig. 8, the reporter gene activity was not significantly altered by hypoxic treatment (1%> O 2 ) in the absence of coexpressed proteins, most probably due to the low levels of endogenous HF-1 activity in COS 7 cells (unpublished observations).
  • pT81/HRE-luc reporter gene activity was stimulated around 10-fold under normoxic conditions, possibly due to a small but detectable pool of HF-l ⁇ localized in the nucleus under these conditions.
  • This reporter gene activity showed a modest increase ( ⁇ 1.5-fold) by hypoxia (Fig. 8).
  • ectopic expression of CBP further enhanced hypoxia-dependent activation by HF- l ⁇ in a dose-dependent manner when compared with hypoxia stimulated activity in the absence of exogenous coactivators (2- to 3-fold).
  • expression of CBP slightly stimulated HF-l activity already at normoxia, its overall activity was almost entirely dependent on the exposure to hypoxia as has been described previously.
  • TF-2 which was originally identified as a coactivator supporting hormone- dependent transcriptional activation by members of the steroid hormone receptor family also functions as a conditionally regulated coactivator in hypoxia-dependent transcriptional activation by the HF-l ⁇ / Arnt complex.
  • HF-l ⁇ In response to hypoxia, HF-l ⁇ is imported to the nucleus and shows a strictly nuclear localization. To characterize the mechanism of TF2-dependent enhancement of transcriptional activation by HF-l ⁇ in living cells we next asked whether overexpression of TF2 would affect the intracellular localization of HF-l ⁇ . To this end we transiently transfected COS7 cells with an expression vector encoding an in-frame fusion of green fluorescent protein (GFP) with full-length HF-l ⁇ .
  • GFP green fluorescent protein
  • TF2 is a nuclear protein mainly associated with such dot-like discrete bodies within nucleus (Voegel et al. (1998) EMBO J. 17, 507-519). These observations strongly suggest that TF-2 induced relocalization of GFP- HF-l ⁇ within the nucleus, indicating that GFP- HF-l ⁇ and TF2 interacted with one another in vivo, and that the dot-like fluorescence pattern might represent transcriptionally active chromatin.
  • TF2 belongs to a growing family of the pi 60 family of coactivators including SRC-1. Both TF2 and SRC-1 appear to have similar activities as coactivators to enhance the transcriptional potential of many members of the ligand-activated nuclear hormone receptors (Voegel et al., supra). SRC-1 has been demonstrated to directly and constitutively interact with CBP (Yao et al. (1996) Proc. Natl. Acad. Sci. USA 93, 10626- 10631). Moreover, in analogy to CBP, SRC-1 has been shown to possess intrinsic histone acetyltransferase activity (Spencer et al. (1997) Nature 389, 194-198).
  • Fig. 9C the pi 60 family of coactivators belongs to the larger family of bHLH/PAS factors (schematically represented in Fig. 9C).
  • SRC-1 was originally cloned as an N-terminally truncated fragment (Onate et al. (1995) Science 270, 1354-1357), here termed SRC-I ⁇ PAS (Fig. 9C), lacking the bHLH/PAS motifs.
  • Both the bHLH and PAS domains represent potent protein/protein interaction interfaces, and are critical for dimerization between HF-l ⁇ and Arnt (Gradin et al. (1996) Mol. Cell Biol. 16, 5221-5231; Wang et al. (1995) Proc.
  • SRC-1 contains four copies of the short sequence motif, LXXLL. Three of these motifs were identified in the central interaction domain of the protein, and the fourth motif was found in the eight most C-terminal amino acids of human SRC-1 (Heery et al. (1997) Nature 387, 733- 736). The LXXLL motifs have been shown to be necessary to mediate the binding of SRC-1 o to ligand occupied nuclear receptors.
  • GAL4/HF 71-826 which lacks the bHLH domain of HF-l ⁇ (The amino acid sequence of HF-l ⁇ /71-826 is shown as SEQ ID NO: 11), induced relative luciferase activity about 3.5- fold over background levels (Fig. 11 A).
  • luciferase activity was induced under hypoxic conditions by GAL4/HF 71-826 to 30- and 34-fold higher levels, respectively, in comparison with the activity observed at hypoxia in the absence of exogenous coactivators.
  • residues 813-826 a region that is contained within the C-terminal transactivation domain of HF-l ⁇ and has been identified as a point of interaction with CBP (Arany et al. (1996) Proc. Natl. Acad. Sci. USA 93, 12969- 12973).
  • GAL4/HF 1-813 The amino acid sequence of HF-l ⁇ /1-813 is shown as SEQ ID NO: 12
  • SEQ ID NO: 12 maintained a hypoxia-inducible response that was significantly enhanced (14- to 13-fold) by overexpression of CBP and SRC-1 (Fig. 11 A), indicating that the hypoxia-dependent function of the N-terminal transactivation domain of HF-l ⁇ may also be regulated by the coactivators.
  • GAL4 HF 531-584 (The amino acid sequence of HF-l ⁇ /531-584 is shown as SEQ ID NO: 13), a chimeric protein spanning the N-terminal transactivation domain of HF-l ⁇ , showed hypoxia- dependent induction of reporter gene activity, albeit with a lower potency than the constructs spanning both transactivation domains.
  • CBP C-binding protein
  • SRC-1 SRC-1 only slightly (1.5-fold) enhanced the activity of GAL4/HF 531-584 under identical conditions.
  • a fusion protein containing the carboxy-terminal transactivation domain of HF-l ⁇ , GAL4/HF 776-826 (The amino acid sequence of HF-l ⁇ /776-826 is shown as SEQ ID NO: 14), produced a very modest (about 2-fold) hypoxia-dependent induction response. However, its transcriptional activity was dramatically enhanced in the presence of either CBP (29-fold increase) or SRC-1 (17-fold increase).
  • CBP CBP
  • SRC-1 SRC-1
  • Arnt the functional partner factor of HF-l ⁇
  • CBP/p300 Kobayashi et al. (1997) J. Biochem. 122, 703-710) we also examined whether SRC-1 might functionally interact with Arnt.
  • Arnt functions as a constitutively active transcription factor on E box-driven promoters.
  • COS7 cells Arnt fused to the GAL4 DBD together with either CBP or SRC- 1.
  • constitutive activation of the reporter gene by GAL4/Arnt 128-774 was potently further enhanced by coexpression of CBP or SRC-1 (Fig. 1 IB).
  • GAL4 fusion proteins spanning the isolated transactivation domains showed an additive mode of regulation in the presence of both SRC- 1 and CBP, strongly suggesting that synergistic regulation by these two coactivators may require the presence and integrity of both transactivation domains of HF-l ⁇ .
  • CBP and p300 are closely related proteins that exhibit strong sequence similarity and similar functions with respect to their roles as coactivators (Arany et al. (1994) Cell 77, 799-800).
  • p300 enhanced in the presence of SRC-1 hypoxia-dependent transcriptional activation of reporter genes by GAL4 fusion proteins containing either one of the two individual transactivation domains of HF-l ⁇ (Fig. 12C).
  • the CBP/p300 and SRC-1 classes of coactivators produced together rather modest (3- to 4-fold) hypoxic activation of the N-terminal transactivation domain located between residues 531 and 584 of HF-l ⁇ (Fig. 12C), whereas, in the presence of SRC-1, CBP or p300 more potently (around 11 -fold) stimulated hypoxic activation by the C-terminal transactivation domain of HF-l ⁇ (Fig. 12C).
  • p300 ⁇ HAT was about 2-fold less potent than wild-type p300 in producing this response (Fig. 12C), indicating that the acetyltransferase catalytic activity of p300 may contribute to trigger the full activity of the HF-l ⁇ -mediated transcriptional activation response.
  • SRC-1 also harbors intrinsic histone acetyltransferase activity (Spencer et al. (1997) Nature 389, 194-198)
  • the effect of the deletion of the acetyltransferase domain of p300 may be masked by corresponding activities of CBP-associated proteins, possibly that of SRC-1 itself.
  • Ref-1 potentiates HIF-l ⁇ function in the presence of CBP and SRC-1
  • the nuclear redox regulator Ref-1 is known to stabilize the DNA binding activity of AP-1 by reduction of a conserved cysteine residue of Fos and Jun.
  • Ref-1 is a bifunctional enzyme: it harbors both redox and endonuclease DNA repair activities (Xanthoudakis et al. (1992) EMBO J. 11, 3323-3335) and has been implicated in up-regulation of HF-l ⁇ -dependent induction of gene expression under hypoxic conditions (Huang et al. (1996) J. Biol. Chem. 271, 32253-32259).
  • hypoxia-inducible transcriptional potencies of all the fusion proteins containing either both or the individual HF-l ⁇ transactivation domains were dramatically (10- to 53-fold) enhanced by coexpression of Ref-1 in the presence of CBP and SRC-1 (Fig. 13B).
  • the fusion proteins spanning the N- or C-terminal transactivation domains produced 12- or 53-fold hypoxia-dependent activation responses, respectively, whereas the fusion protein containing the C-terminal transactivation domain lacking the CBP interaction interface, GAL4/HF 776-813, showed no regulation by Ref-1 and the coactivators.
  • Ref-1 activates HF-l ⁇
  • a GFP- tagged Ref-1 fusion protein was exclusively localized in the nucleus both under normoxic and hypoxic conditions indicating that nuclear translocation of HF-l ⁇ is required for functional interaction with Ref-1.
  • This redox regulator protein has been shown to form complexes with Jun in vitro (Xanthoudakis et al. (1994) Proc. Natl. Acad. Sci. USA 91, 23- 27).
  • a crosslinking reagent such as diamide, which oxidizes cysteine sulfhydryls to disulfides.
  • auxiliary factor possibly a coactivator, may mediate interaction with at least the N-terminal transactivation domain of HF-l ⁇ .
  • pCMX-SAH/Y145F expression vector encoding a modified and highly chromophoric form of GFP under the control of cytomegalovirus (CMV) immediate early promoter was a generous gift from Dr. Kazuhiko Umesono (Kyoto University, Japan).
  • CMV cytomegalovirus
  • This humanized GFP contains a S65A mutation which confers a wavelength shift and temperature resistance to the protein as well as a Y145P substitution increasing the intracellular stability of GFP.
  • pRc/RSV-mCBP-HA construct expressing full-length mouse CBP was a gift from Dr. Richard H. Goodman (Vollum Institute, Oregon).
  • the vector expressing a GFP-fusion of full-length HF-l was generated by cutting the HF-l ⁇ coding region from pGEX-4T3-HF- l ⁇ as a BamHVNot I fragment (Notl site filled-in with Klenow polymerase) and ligating this in-frame into BamHUNhel opened pCMX-SAH7Y145F where the Nhel site had been blunt- ended with Klenow.
  • Deletion mutant HF-1 ⁇ /1 -652 was assembled by inserting a BamHUSpel fragment of HF-l ⁇ into £a/wHI/ eI-digested pCMX-SAH/Y145F.
  • GFP fusions encoding HF-l ⁇ subfragments were generated by amplifying the equivalent D ⁇ A sequences by PCR using Pfu D ⁇ A polymerase (Stratagene) together with primer pairs carrying BamHl or Nhel ends. The resulting products were inserted into BamHUNhel opened pCMX-SAH/Y145F.
  • GFP fusions carrying amino terminal sequences HF-l ⁇ (1-74, 1-245, 1-330) were cut out from corresponding bacterial GST fusion expression vectors and inserted in frame to BamHUNhel site (Nhel blunt-ended) of pCMX-SAH/Y145F.
  • GFP-HF-l ⁇ fusion proteins ending at amino acid 813 were generated by cleaving the C-terminal 13 amino acids by Pstl digestion.
  • Corresponding GAL4 D ⁇ A-binding domain fusion proteins were assembled by cleaving the HF-l ⁇ inserts as Bam ⁇ llNhel fragments and religating them into pCMX expression vector.
  • Site-directed mutagenesis of the C-terminal ⁇ LS was performed by overlap PCR (Ausubel et al, 1994) where the desired mutation (codon 719 AAG ->ACA was introduced into a PCR product and then inserted as an EcoRl-Pstl subfragment into pGFP-HF-l ⁇ /526-826.
  • a GFP fusion of full-length HF- 1 ⁇ carrying K719T mutation was thereafter assembled by inserting the aminoterminal BamEl-Spel fragment of HF-l ⁇ into pGFP-HF-l ⁇ /526-826 K719T.
  • PCR-based mutagenesis with specific oligonucleotides was employed to generate mutations to the ⁇ - terminal ⁇ LS motif. Fidelity of PCR reactions and identity of constructs was confirmed by sequencing the inserts.
  • COS7 cells (from ATCC) were routinely maintained in Dulbecco's minimal essential medium (DMEM) supplemented with 10%> fetal calf serum plus penicillin (50 IU/ml) and streptomycin (50 ⁇ g/ml).
  • DMEM Dulbecco's minimal essential medium
  • penicillin 50 IU/ml
  • streptomycin 50 ⁇ g/ml
  • GFP-tagged HF-l ⁇ and its various mutant plasmids in COS7 cells The cells were cultured on the silane-coated cover slips in 6-cm diameter plastic dishes, and the medium was changed to OPTI-MEM medium lacking phenol red (Life Technologies, Inc.) before transfection.
  • a plasmid cocktail containing 6 ⁇ g of the expression plasmids for GFP- tagged HF-l ⁇ and its various mutants was mixed with 12 ⁇ l of TransIT-LTl reagent (Panvera Corp., Madison, WI) and added to the culture. After 6 h of incubation, the medium was replaced with DMEM with 10% FCS. Cells were induced 24 h later with 1% O 2 , 100 ⁇ M 2,2'-dipyridyl or 100 ⁇ M C0CI2 or treated with vehicle only.
  • GFP and GAL4 fusion constructs were analyzed in a cotransfection assay where effector plasmids (0.2 ⁇ g/ 30-mm dish) together with a reporter gene (1.5 ⁇ g/ 30-mm dish) were introduced into COS7 or HeLa cells.
  • the reporter plasmid encoded firefly luciferase gene under the control of thymidine kinase minimal promoter and either five or three copies of GAL4 or HF-responsive elements, respectively.
  • COS7 cells grown on fibronectin-coated cover-slips were transiently transfected with the expression plasmid for full-length HF-l ⁇ (pCMN-HF-l ⁇ ) or HF-l ⁇ fused to the GAL4 D ⁇ A binding domain (pCMX-GAL4-HF-l ⁇ ). After various treatments, the cells were fixed with 4% paraformaldehyde in PBS at room temperature for 30 min. Immunostaining of the cells was carried out using anti-HF-l ⁇ antiserum (Kallio et al. (1997) Proc. ⁇ atl. Acad. Sci.
  • Transiently expressed GFP was expressed at detectable levels between 24 and 72 h after transfection. Routinely, cells were used for further experiments 48 h after transfection. After various treatments, cells were examined using a Zeiss Axiovert 135 microscope enclosed by an incubator and equipped with a heating-stage, an FITC-filter set. and epifluorescence with illumination from a Gixenon burner (Carl Zeiss Jena GmbH, Jena, Germany). Photographs were taken using Kodak Echtachrome 400, and semi-quantitative assessment of the subcellular lo;alization of the GFP-tagged proteins was performed according to the methods described by Ylikomi et al. ((1992) EMBO J. 11, 3681-3694) .
  • the cell pellet was frozen in liquid nitrogen and thawed by resuspending in 80 ⁇ l of cell extraction buffer (10 mM Hepes, pH 7.9; 400 mM NaCl; 0.1 mM EDTA; 5% (v/v) glycerol; 1 mM dithiotreitol; 1 mM phenylmethylsulfonyl fluoride), followed by centrifugation for 30 min at maximal velocity. Fifty ⁇ g of the total cell proteins were blotted after SDS polyacrylamide gel electrophoresis onto nitrocellulose filter and blocked overnight with 5 %> nonfat milk in PBS.
  • cell extraction buffer 10 mM Hepes, pH 7.9; 400 mM NaCl; 0.1 mM EDTA; 5% (v/v) glycerol; 1 mM dithiotreitol; 1 mM phenylmethylsulfonyl fluoride
  • Anti-H -l ⁇ antiserum was used as a primary antibody as 1 :500 dilution in PBS containing 1%> nonfat milk for 2 hours. After washes, 1 :750 dilution of anti-rabbit IgG- horseradish peroxidase conjugate (Amersham) in PBS / 1 % nonfat milk was used as a secondary antibody. After extensive washing with PBS the complexes were visualized using enhanced chemiluminescence (Amersham) according to the manufacturer's instructions. TABLE I
  • N cells containing exclusively nuclear fluorescence
  • N>C cells in which the nuclear fluorescence exceeds the cytoplasmic fluorescence
  • N ⁇ C cells having cytoplasmic fluorescence exceeding that in the nucleus.

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Abstract

L'invention porte sur un procédé d'identification de composés susceptibles de moduler la fonction d'un domaine fonctionnel du HIF-1α humain ledit procédé consistant (i) à mettre en contact un composé candidat avec un variant du HIF-1α humain, ledit variant manquant au moins d'un domaine fonctionnel du HIF-1α humain, ou présentant une mutation rendant quasiment inactif l'un au moins des domaines du HIF-1α humain, le ou lesdits domaines fonctionnels étant sélectionnés parmi: (a) le domaine PAS-B situé dans le HIF-1α humain entre les acides aminés 178 et 390, (b) la séquence C-terminale (NLS) de localisation du noyau situé dans le HIF-1α humain essentiellement au niveau des acides aminés 718 à 721, et (c) le domaine transactivateur / coactivateur (N-TAD) situé dans le HIF-1α humain essentiellement entre les acides aminés 531 à 584, et (d) le domaine transactivateur / coactivateur (C-TAD) situé dans le HIF-1α humain essentiellement entre les acides aminés 813 et 826, puis (ii) à déterminer l'effet dudit composé candidat sur ledit variant.
PCT/SE1999/002053 1998-11-13 1999-11-11 VARIANTS HIF-1α DU FACTEUR INDUCTIBLE PAR L'HYPOXIE (HIF), LEURS METHODES D'IDENTIFICATION, ET LEURS MODULATEURS WO2000029437A1 (fr)

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JP2000582422A JP2002530063A (ja) 1998-11-13 1999-11-11 低酸素誘導性因子１α（ＨＩＦ−１α）変異体およびＨＩＦ−１α調整因子の同定法
EP99958594A EP1141005B1 (fr) 1998-11-13 1999-11-11 Methodes d'identification de modulateurs du facteur inductible par l'hypoxie hif-1alpha
CA002350830A CA2350830A1 (fr) 1998-11-13 1999-11-11 Variants hif-1.alpha. du facteur inductible par l'hypoxie (hif), leurs methodes d'identification, et leurs modulateurs
NZ511450A NZ511450A (en) 1998-11-13 1999-11-11 Method of identifying compounds that modulate the function of the human hypoxia-inducible factor 1-alpha (HIF-1alpha) functional domain
DE69937199T DE69937199D1 (de) 1998-11-13 1999-11-11 Verfahren zur identifikation von modulatoren von hif-1-alpha
BR9915263-0A BR9915263A (pt) 1998-11-13 1999-11-11 Variantes do fator hif-1 "alfa" que induz à hipoxiahumana e métodos para identificação dosmoduladores de hif-1 "alfa"
AU15931/00A AU761594B2 (en) 1998-11-13 1999-11-11 Hypoxia-inducible factor 1alpha HIF-1alpha variants and methods for identifying HIF-1alpha modulators
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EP1779860A2 (fr) * 2005-10-26 2007-05-02 Chengqiu Wang Peptides de chromatine bloquant le gène HIF-1 alpha et modulant les gènes situés en aval
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US6787326B1 (en) 1999-05-12 2004-09-07 Isis Innovation Limited Interaction between the VHL tumor suppressor and hypoxia inducible factor, and assay methods relating thereto
WO2000069908A1 (fr) * 1999-05-12 2000-11-23 Isis Innovation Limited Interaction entre le suppresseur de tumeurs de la maladie de von hippel-lindau (vhl) et le facteur induit par l'hypoxie (hif) et procedes de dosage qui y sont lies
EP1198255A4 (fr) * 1999-06-04 2005-06-15 Dana Farber Cancer Inst Inc Identification de composes qui modifient les reponses transcriptionnelles a l'hypoxie
EP1198255A1 (fr) * 1999-06-04 2002-04-24 Dana-Farber Cancer Institute, Inc. Identification de composes qui modifient les reponses transcriptionnelles a l'hypoxie
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WO2003064587A2 (fr) * 2002-01-11 2003-08-07 Angio Genetics Sweden Ab Repetitions de di-leucine dans le domaine c-terminal de transactivation du facteur alpha 1 inductible par l'hypoxie, critiques pour la transactivation, interaction de coactivateur et trafic intranucleaire, produits et procedes associes
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ZA200103669B (en) 2002-05-07
CN1330666C (zh) 2007-08-08
BR9915263A (pt) 2001-10-30
AU761594B2 (en) 2003-06-05
EP1141005A1 (fr) 2001-10-10
NZ511450A (en) 2003-08-29
EP1141005B1 (fr) 2007-09-26
CA2350830A1 (fr) 2000-05-25
HK1043372B (zh) 2008-05-09
HK1043372A1 (en) 2002-09-13
AU1593100A (en) 2000-06-05
ATE374210T1 (de) 2007-10-15
DE69937199D1 (de) 2007-11-08
CN1330663A (zh) 2002-01-09
KR20010080411A (ko) 2001-08-22
SE9803891D0 (sv) 1998-11-13
KR100648761B1 (ko) 2006-11-23
TR200101345T2 (tr) 2001-10-22

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